The invention relates to a process for reacting a carboxylic acid ester of the formula (I)R1-A-COOR2  (I),
wherein R1 is selected from the group comprising hydrogen, —CH2OH, —CHO, —COOR3, —CH2SH, —CH2OR3 and —CH2NH2,
wherein R2 is selected from the group comprising hydrogen and alkyl, preferably methyl, ethyl and propyl,
wherein R3 is selected from the group comprising hydrogen and alkyl, preferably methyl, ethyl and propyl,
and wherein A is a substituted, unsubstituted, linear, branched and/or cyclic alkylene, alkenylene, arylene or aralkylene radical having at least 4, more preferably 6, even more preferably 8, carbons,
by means of a cell, comprising the step of
a) contacting the cell with said carboxylic acid ester in an aqueous solution,
wherein the cell is a recombinant cell which has reduced activity of a polypeptide having SEQ ID NO: 2 or a variant thereof over the wild-type cell, and to a cell suitable therefor, and to uses thereof.
Biotechnology is concerned with producing inter alia fine chemicals, employing various organisms that possess interesting synthesizing capabilities. Biotechnological processes have a number of advantages over conventional chemical processes. Firstly, they usually dispense entirely with harmful substances such as heavy metal-based catalysts and reaction conditions involving extreme pH, pressure and temperature values. Furthermore, the infrastructure required for the biotechnological process can often be set up with lower costs and safety measures. Selectivity and specificity of biotechnologically relevant enzymes often exceed those of chemical catalysts considerably, and formation of undesired secondary products which frequently are difficult to remove from the product and which would necessarily be produced in a synthesis using organic synthesis processes can thus be reduced. Finally, biotechnologically relevant organisms in many cases accept as reactants compounds like complex carbohydrates which may be derived from renewable raw materials. As a result, a producing enterprise may reduce its dependence on fossil raw materials such as petroleum and natural gas.
Establishing biotechnological processes, however, entails considerable difficulties which result in only very few substances being biotechnologically produced on an industrial scale these days, the main problem being that a cell with a desired synthesizing activity has not only the one enzyme responsible for said synthesizing activity but rather thousands of enzymes which co-exist in the same cell and compete with one another for substrates or even catalyse completely opposing reactions. Thus in the genome of Escherichia coli alone, approx. 80 polypeptides are encoded which have been identified by bioinformatics methods as hydrolases, i.e. enzymes which cleave particular bonds with consumption of a water molecule. Indeed, the conditions under which an enzyme is produced by the cell and the reactions catalysed by said enzyme and the substrates involved in said reactions have been elucidated exhaustively only in a few cases. It is therefore in many cases not possible to specifically select an enzyme for catalysing a particular reaction.
Accordingly, when employing cells as biocatalysts rather than chemical or isolated biological catalysts, there is also always the risk of a product or intermediate produced by an enzyme equipped with a desired activity or even the original reactant being converted by another enzyme into an unwanted secondary product. Whether this will happen and which of the numerous enzymes will have said unwanted activity in this case is impossible to predict in spite of technical advances in the field of bioinformatics.
It is not unlikely, especially with chemically reactive substances desired in the industry as reactive reactants for producing more complex products, that said substances react inside the cell with essential components of the organism and thus have a toxic action. If this is the case, the ability of the organism to grow and synthesize will be impaired, and ultimately the cell will die, without the developer being able to immediately recognize said toxicity. Likewise, the organism that will tolerate a chemically reactive substance as well as the tolerated concentration of the latter cannot be predicted.
In processes involving a plurality of reactions catalysed in each case by an enzyme, the complexity of the system makes the search for factors limiting yield or purity more difficult. If the product yield is too low, the reason for this may be the concentration of one of the enzymes present being too low, although that enzyme would not be known from among possible enzymes, that is to say owing to insufficient synthesizing capacity, the reactant is not reacted within the intended time frame or prior to degradation by competing enzymes. Alternatively, it is quite possible that an enzyme, although detectably present as a polypeptide in the cell, does not have the folding essential to the activity in that particular cell or that a hitherto unknown cofactor which is, however, essential to the activity is missing. Equally, as mentioned previously, the metabolic product may be toxic to the cell or broken down.
A person skilled in the art who would like to establish or improve a biotechnological process is thus confronted with numerous possible starting points, but in most cases is not provided by the prior art with any specific and executable instruction as to which of these starting points he needs to start from in order to achieve the objective.
Carboxylic acid esters constitute a group of compounds that are in high demand in the industry and that, either themselves or by way of processed products, are used as pharmaceuticals, cosmetics, plastics and the like.
Processing, however, frequently requires not only an ester function, which has to be introduced into a precursor first, but also further derivatization being carried out on the ester without hydrolysing the ester function in the process or subsequently. The latter is not a trivial accomplishment to be achieved, since many carboxylic acid esters tend to hydrolyse even in the absence of enzymes catalysing such reactions, in particular in aqueous solutions and at pH values greatly deviating from the neutral point.
An important possibility of further derivatizing carboxylic acid esters is oxidization of alkyl chains present therein. This first produces an alcohol which either is used as such or may be further oxidized to the aldehyde or ketone. The aldehyde or ketone may be either reductively aminated or further oxidized to the carboxylic acid which in turn may be esterified again, if required.
This multiplicity of possible reactions, many of which are catalysed by endogenous enzymes, i.e. enzymes that are naturally present in an organism, indicates that the problem of uncontrolled secondary product formation or metabolizing is particularly serious for reacting carboxylic acid esters by means of biotechnological processes.
An example of a carboxylic acid ester that is in high demand in the industry and that is customarily prepared starting from hydrocarbons present in petroleum is methyl 12-amino-dodecanoate(ALSME)[12-Aminolauric acid methylester]. ALSME is an important starting material in the preparation of polymers, for example for producing nylon-based line systems. ALSME has previously been produced in a low-yield process, starting from fossil raw materials.
A promising new way of biotechnologically producing ALS or ALSME is described in WO 2009/077461. This involves methyl dodecanoate being oxidized by a monooxygenase in a first step, and reacting the resultant aldehyde by means of a transaminase to give ALSME. A disadvantage of this process is the production of secondary products, for example the dicarboxylic acid, which can be removed from the desired product, ALSME, only with difficulties. This reduces the yield and makes recycling of hydrophobic solvents and hydrophobic cation exchangers, which may be used according to PCT/EP2011/071491 for removing the product from the aqueous reaction mixture, more difficult, at the expense of efficient resource usage.
Against this background, it is an object of the invention to provide a biotechnological process for reacting carboxylic acid esters which is as efficient as possible with regard to yield, carbon balance and/or nitrogen balance and/or purity.
Another object of the invention is to provide a biotechnological process for reacting carboxylic acid esters to give aminated carboxylic acid esters, which is as efficient as possible with regard to yield, carbon balance and/or nitrogen balance, re-usability of agents used and/or purity of the product. In this connection, an efficient carbon balance and/or nitrogen balance preferably means that the proportion of the carbon and/or nitrogen fed in the form of suitable substrates to a cell for reacting a carboxylic acid ester is as high as possible in the desired final product, instead of being reacted to give products other than the desired ones, for example.
Another object of the invention is to improve the ability of a multi-phase reaction mixture to be worked up from the reaction of a carboxylic acid ester, particularly with regard to re-usability of hydrophobic solvents and liquid cation exchangers used for work-up, and with regard to phase formation and separation in a biphasic system comprising an aqueous phase in which reaction of the carboxylic acid ester takes place and an organic phase with organic solvents and/or liquid cation exchangers.